Aberrations in the mitotic apparatus can lead to chromosome nondisjunction and aneuploidy associated with malignancy and human genetic and developmental disorders. This research is designed to elucidate the structure of the mitotic apparatus and factors that regulate the initiation and assembly of spindle microtubules. Chromosome movement in eukaryotic cells requires the regulated assembly and disassembly of spindle microtubules during mitosis. Microtubules of the mitotic apparatus (MA) like those of the cytoplasmic microtubule complex (CMTC) assemble in association with discrete microtubule-organizing centers (MTOCs). The overall objective of this research is to define further the structure and molecular compositions of MTOCs and to elucidate their function in regulating the assembly of specific microtubule arrays during the mitotic cycles. The specific aims are: (1)\to isolate MTOCs (kinetochores and centrosomes) and to demonstrate functional components and molecular composition required for tubulin initiation and polymerization; (2)\to further characterize human autoantibodies to MTOCs for use as cytological and biochemical probes; (3)\to prepare monoclonal antibodies to MTOCs for use as cytological and biochemical probes; (4)\to continue to analyze MTOCs in living cells and lysed cell models; and (5)\to analyze new taxol-requiring mutant CHO cells that fail to form a metaphase spindle and appear to have defective MTOCs. This project will be carried out on a number of transformed and nontransformed cell lines including mouse neuroblastoma, Chinese hamster ovary (CHO), 3T3, and SV3T3 cells. (L)